194 results on '"Osaki, Toshihisa"'
Search Results
152. MNM-1A-3 A Device for Size Separation of Vesicles Using Microfluidic Force
- Author
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Yamamoto, Ryosuke, primary, Sasaki, Hirotaka, additional, Osaki, Toshihisa, additional, Kawano, Ryuji, additional, Miki, Norihisa, additional, and Takeuchi, Shoji, additional
- Published
- 2010
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153. A parylene nanopore for stable planar lipid bilayer membranes
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Kawano, Ryuji, primary, Osaki, Toshihisa, additional, and Takeuchi, Shoji, additional
- Published
- 2010
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- View/download PDF
154. Electro-Optical Imaging Microscopy of Dye-Doped Artificial Lipidic Membranes
- Author
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Hajj, Bassam, primary, De Reguardati, Sophie, additional, Hugonin, Loïc, additional, Le Pioufle, Bruno, additional, Osaki, Toshihisa, additional, Suzuki, Hiroaki, additional, Takeuchi, Shoji, additional, Mojzisova, Halina, additional, Chauvat, Dominique, additional, and Zyss, Joseph, additional
- Published
- 2009
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155. Multichannel Simultaneous Measurements of Single-Molecule Translocation in α-Hemolysin Nanopore Array
- Author
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Osaki, Toshihisa, primary, Suzuki, Hiroaki, additional, Le Pioufle, Bruno, additional, and Takeuchi, Shoji, additional
- Published
- 2009
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- View/download PDF
156. Electrical recording of lipid membrane in a microfluidic device
- Author
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Watanabe, Yoshihiko, primary, Osaki, Toshihisa, additional, and Takeuchi, Shoji, additional
- Published
- 2009
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157. Electro-Optical Imaging Microscopy of Dye Doped Lipid Bilayer
- Author
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HAJJ, Bassam, primary, DE REGUARDATI, Sophie, additional, HUGONIN, Loïc, additional, Osaki, Toshihisa, additional, Suzuki, Hiroaki, additional, Takeuchi, Shoji, additional, LE PIOUFLE, Bruno, additional, CHAUVAT, Dominique, additional, and ZYSS, Joseph, additional
- Published
- 2009
- Full Text
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158. 3P-208 Transport Property of Polynucleotide Molecules on α-Hemolysin Nanopore Array(The 46th Annual Meeting of the Biophysical Society of Japan)
- Author
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Osaki, Toshihisa, primary, Suzuki, Hiroaki, additional, Pioufle, Bruno Le, additional, and Takeuchi, Shoji, additional
- Published
- 2008
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159. Combined microslit electrokinetic measurements and reflectometric interference spectroscopy to study protein adsorption processes
- Author
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Zimmermanna, Ralf, primary, Osaki, Toshihisa, additional, Gauglitz, Günter, additional, and Werner, Carsten, additional
- Published
- 2007
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160. Nonlinear concentration gradients regulated by the width of channels for observation of half maximal inhibitory concentration (IC50) of transporter proteins.
- Author
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Abe, Yuta, Kamiya, Koki, Osaki, Toshihisa, Sasaki, Hirotaka, Kawano, Ryuji, Miki, Norihisa, and Takeuchi, Shoji
- Subjects
CARRIER proteins ,INHIBITORY Concentration 50 ,MICROFLUIDIC devices ,CONCENTRATION gradient ,ATP-binding cassette transporters ,DRUGS - Abstract
This paper describes a simple microfluidic device that can generate nonlinear concentration gradients. We changed the “width” of channels that can drastically shorten the total microfluidic channel length and simplify the microfluidic network design rather than the “length” of channels. The logarithmic concentration gradients generated by the device were in good agreement with those obtained by simulation. Using this device, we evaluated a probable IC
50 value of the ABC transporter proteins by the competitive transport assays at five different logarithmic concentrations. This probable IC50 value was in good agreement with an IC50 value (0.92 μM) obtained at the diluted concentrations of seven points. [ABSTRACT FROM AUTHOR]- Published
- 2015
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161. Polyanion Protection of Silane Bonds to Silicon Oxide Revealed by Electrokinetic Measurements
- Author
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Osaki, Toshihisa, primary, Zimmermann, Ralf, additional, Kratzmüller, Thomas, additional, Schweiss, Rüdiger, additional, and Werner, Carsten, additional
- Published
- 2003
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162. Ionization Characteristics and Structural Transitions of Alternating Maleic Acid Copolymer Films
- Author
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Osaki, Toshihisa, primary and Werner, Carsten, additional
- Published
- 2003
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163. Dielectric Relaxation on the Intermediate Layer in a Bipolar Membrane under the Water Splitting Phenomenon
- Author
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Osaki, Toshihisa, primary and Tanioka, Akihiko, additional
- Published
- 2002
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164. Effects of pH on the Transport of 5-Fluorouracil across a Fibroin Membrane.
- Author
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Chen, Jianyong, primary, Minoura, Norihiko, additional, Osaki, Toshihisa, additional, and Tanioka, Akihiko, additional
- Published
- 2000
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165. Parallel Recordings of Transmembrane hERG Channel Currents Based on Solvent-Free Lipid Bilayer Microarray.
- Author
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Miyata, Ryusuke, Tadaki, Daisuke, Yamaura, Daichi, Araki, Shun, Sato, Madoka, Komiya, Maki, Ma, Teng, Yamamoto, Hideaki, Niwano, Michio, Hirano-Iwata, Ayumi, and Osaki, Toshihisa
- Subjects
DRUG side effects ,PHARMACOLOGY ,ION analysis ,LIPIDS ,FUNCTIONAL analysis ,BILAYER lipid membranes ,ION channels - Abstract
The reconstitution of ion-channel proteins in artificially formed bilayer lipid membranes (BLMs) forms a well-defined system for the functional analysis of ion channels and screening of the effects of drugs that act on these proteins. To improve the efficiency of the BLM reconstitution system, we report on a microarray of stable solvent-free BLMs formed in microfabricated silicon (Si) chips, where micro-apertures with well-defined nano- and micro-tapered edges were fabricated. Sixteen micro-wells were manufactured in a chamber made of Teflon
® , and the Si chips were individually embedded in the respective wells as a recording site. Typically, 11 to 16 BLMs were simultaneously formed with an average BLM number of 13.1, which corresponded to a formation probability of 82%. Parallel recordings of ion-channel activities from multiple BLMs were successfully demonstrated using the human ether-a-go-go-related gene (hERG) potassium channel, of which the relation to arrhythmic side effects following drug treatment is well recognized. [ABSTRACT FROM AUTHOR]- Published
- 2021
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166. CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6.
- Author
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Maruyama, Yohei, Sugawa, Mitsuhiro, Yamaguchi, Shin, Davies, Tim, Osaki, Toshihisa, Kobayashi, Takuya, Yamagishi, Masahiko, Takeuchi, Shoji, Mishima, Masanori, and Yajima, Junichiro
- Subjects
- *
CYTOKINESIS , *KINESIN , *MICROTUBULES , *MONOMERS , *CAENORHABDITIS elegans - Abstract
Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules. Analysing the 3D movement of MKLP1 motors, Maruyama et al. find that dimeric C. elegans MKLP1 drives a left-handed helical motion around the microtubule with minimum protofilament switching to the right side whereas less persistent motions are driven by monomers or by heterotetramers with CYK4. These findings suggest how obstacles along crowded spindle microtubules may be avoided by CYK4 binding to MKLP1. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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167. Open-source and low-cost miniature microscope for on-site fluorescence detection.
- Author
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Kawai M, Oda H, Mimura H, Osaki T, and Takeuchi S
- Abstract
The development of a compact and affordable fluorescence microscope can be a formidable challenge for growing needs in on-site testing and detection of fluorescent labeled biological systems, especially for those who specialize in biology rather than in engineering. In response to such a situation, we present an open-source miniature fluorescence microscope using Raspberry Pi. Our fluorescence microscope, with dimensions of 19.2 × 13.6 × 8.2 cm
3 (including the display, computer, light-blocking case, and other operational requirements), not only offers cost-effectiveness (costing less than $500) but is also highly customizable to meet specific application needs. The 12.3-megapixel Raspberry Pi HQ Camera captures high-resolution imagery, while the equipped wide-angle lens provides a field of view measuring 21 × 15 mm2 . The integrated wireless LAN in the Raspberry Pi, along with software-controllable high-powered fluorescence LEDs, holds potential for a wide range of applications. This open-source fluorescence microscope offers biohybrid sensor developers a versatile tool to streamline unfamiliar mechanical design tasks and open new opportunities for on-site fluorescence detections., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)- Published
- 2024
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168. Lipid Bilayer Reformation Using the Wiping Blade for Improved Ion Channel Analysis.
- Author
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Mimura H, Osaki T, Takamori S, Nakao K, and Takeuchi S
- Subjects
- Water, Nanotechnology, Lipid Bilayers, Ion Channels
- Abstract
The measurement of ion permeation activity across planar lipid bilayers is a useful technique for the functional analysis and drug evaluation of ion channels at the single-molecule level. To enhance the data throughput, parallelization of lipid bilayers is desirable. However, existing parallelized approaches face challenges in simultaneously and efficiently measuring ion channel activities under various conditions on one chip. In this study, we propose an approach to overcome these limitations by developing a device capable of repeated measurements of ion channels incorporated into individually arrayed lipid bilayers. Our device forms an array of a lipid bilayer at a micropore on a separator by merging two lipid monolayers assembled on the surface of aqueous droplets. We introduce a vertically moving, blade-shaped module─referred to as a "wiping blade"─which enables controlled disruption and reformation of the bilayer at the micropore. By optimizing the surface properties and clearance of the wiping blade, we successfully achieved repeated bilayer formation. The arrayed lipid bilayer device with the integrated wiping blade module demonstrates a 5-fold improvement in data throughput during ion channel activity measurements. Finally, we validate the practical utility of our device by evaluating the effects of an ion channel inhibitor. The developed device opens new avenues for high-throughput analysis and screening of ion channels, leading to significant advancements in drug discovery and functional studies of membrane proteins. It offers a powerful tool for researchers in the field and holds promise for accelerating drug development by targeting ion channels.
- Published
- 2023
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169. Real-time quantitative characterization of ion channel activities for automated control of a lipid bilayer system.
- Author
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Ogishi K, Osaki T, Mimura H, Hashimoto I, Morimoto Y, Miki N, and Takeuchi S
- Subjects
- Humans, Ion Channels, Automation, Lipid Bilayers, Biosensing Techniques
- Abstract
This paper describes a novel signal processing method to characterize the activity of ion channels on a lipid bilayer system in a real-time and quantitative manner. Lipid bilayer systems, which enable single-channel level recordings of ion channel activities against physiological stimuli in vitro, are gaining attention in various research fields. However, the characterization of ion channel activities has heavily relied on time-consuming analyses after recording, and the inability to return the quantitative results in real time has long been a bottleneck to incorporating the system into practical products. Herein, we report a lipid bilayer system that integrates real-time characterization of ion channel activities and real-time response based on the characterization result. Unlike conventional batch processing, an ion channel signal is divided into short segments and processed during the recording. After optimizing the system to maintain the same characterization accuracy as conventional operation, we demonstrated the usability of the system with two applications. One is quantitative control of a robot based on ion channel signals. The velocity of the robot was controlled every second, which was around tens of times faster than the conventional operation, in proportion to the stimulus intensity estimated from changes in ion channel activities. The other is the automation of data collection and characterization of ion channels. By constantly monitoring and maintaining the functionality of a lipid bilayer, our system enabled continuous recording of ion channels over 2 h without human intervention, and the time of manual labor has been reduced from conventional 3 h to 1 min at a minimum. We believe the accelerated characterization and response in the lipid bilayer systems presented in this work will facilitate the transformation of lipid bilayer technology toward a practical level, finally leading to its industrialization., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S. Takeuchi is an inventor of intellectual property rights related to the droplet contact method, and a stockholder of MAQsys Inc., a start-up company that applies the lipid bilayer technology based on the droplet contact method to the validation of drug candidates. T. Osaki is a stockholder and a board member of MAQsys Inc., (Copyright © 2023 Elsevier B.V. All rights reserved.)
- Published
- 2023
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170. 3D printed microfluidic devices for lipid bilayer recordings.
- Author
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Ogishi K, Osaki T, Morimoto Y, and Takeuchi S
- Subjects
- Microfluidics methods, Printing, Three-Dimensional, Stereolithography, Lab-On-A-Chip Devices, Lipid Bilayers
- Abstract
This paper verifies the single-step and monolithic fabrication of 3D structural lipid bilayer devices using stereolithography. Lipid bilayer devices are utilized to host membrane proteins in vitro for biological assays or sensing applications. There is a growing demand to fabricate functional lipid bilayer devices with a short lead-time, and the monolithic fabrication of components by 3D printing is highly anticipated. However, the prerequisites of 3D printing materials which lead to reproducible lipid bilayer formation are still unknown. Here, we examined the feasibility of membrane protein measurement using lipid bilayer devices fabricated by stereolithography. The 3D printing materials were characterized and the surface smoothness and hydrophobicity were found to be the relevant factors for successful lipid bilayer formation. The devices were comparable to the ones fabricated by conventional procedures in terms of measurement performances like the amplitude of noise and the waiting time for lipid bilayer formation. We further demonstrated the extendibility of the technology for the functionalization of devices, such as incorporating microfluidic channels for solution exchangeability and arraying multiple chambers for robust measurement.
- Published
- 2022
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171. Role of Negatively Charged Lipids Achieving Rapid Accumulation of Water-Soluble Molecules and Macromolecules into Cell-Sized Liposomes against a Concentration Gradient.
- Author
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Sugiyama H, Osaki T, Takeuchi S, and Toyota T
- Subjects
- Attention, Membranes, Phospholipids, Liposomes, Water
- Abstract
Liposomes, molecular self-assemblies resembling biological membranes, are a promising scaffold to investigate the physicochemical logic behind the complexity of living cells. Despite elaborate synthetic studies constructing cell-like chemical systems using liposomes, less attention has been paid to the proactive role of the membrane emerging as dynamics of the molecular self-assembly. This study investigated the liposomes containing anionic phospholipids by exposing them to steady flow conditions using a newly constructed automatic microfluidic observation platform. We demonstrated that the liposomes accumulated even macromolecules under the microfluidic condition without pore formation. By investigating the effect of composition of liposomes and visualizing negatively charged phospholipids upon the flow, we presumed that the external flow caused a compositional asymmetry of anionic phospholipids between the inner/outer leaflets, and the asymmetry enabled a rapid accumulation of those molecules against the concentration gradient. The current study opens new research interests regarding the nature of biological membranes under steady flow conditions.
- Published
- 2022
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172. Biohybrid sensor for odor detection.
- Author
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Hirata Y, Oda H, Osaki T, and Takeuchi S
- Subjects
- Odorants, Biosensing Techniques, Receptors, Odorant genetics
- Abstract
Biohybrid odorant sensors that directly integrate a biological olfactory system have been increasingly studied and are suggested to be the next generation of ultrasensitive sensors by taking advantage of the sensitivity and selectivity of living organisms. In this review, we provide a detailed description of the recent developments of biohybrid odorant sensors, especially considering the requisites for their perspective of on-site applications. We introduce the methodologies to effectively capture the biological signals from olfactory systems by readout devices, and describe the essential properties regarding the gaseous detection, stability, quality control, and portability. Moreover, we address the recent progress on multiple odorant recognition using multiple sensors as well as the current screening approaches for pairs of orphan receptors and ligands necessary for the extension of the currently available range of biohybrid sensors. Finally, we discuss our perspectives for the future for the development of practical odorant sensors.
- Published
- 2021
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173. Highly sensitive VOC detectors using insect olfactory receptors reconstituted into lipid bilayers.
- Author
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Yamada T, Sugiura H, Mimura H, Kamiya K, Osaki T, and Takeuchi S
- Abstract
This paper reports a volatile organic compound (VOC) sensor based on olfactory receptors that were reconstituted into a lipid bilayer and used in a specifically designed gas flow system for rapid parts per billion (ppb)-level detection. This VOC sensor achieves both rapid detection and high detection probability because of its gas flow system and array design. Specifically, the gas flow system includes microchannels and hydrophobic microslits, which facilitate both the introduction of gas into the droplet and droplet mixing. We installed this system into a parallel lipid bilayer device and subsequently demonstrated parts per billion-level (0.5 ppb) detection of 1-octen-3-ol in human breath. Therefore, this system extends the various applications of biological odorant sensing, including breath diagnosis systems and environmental monitoring., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)
- Published
- 2021
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174. Efficient Lipid Bilayer Formation by Dipping Lipid-Loaded Microperforated Sheet in Aqueous Solution.
- Author
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Misawa N, Fujii S, Kamiya K, Osaki T, and Takeuchi S
- Abstract
This paper describes a method for a bilayer lipid membrane (BLM) formation using a perforated sheet along with an open chamber. Microscopic observation of the formed membrane showed a typical droplet interface bilayer. We proved that the formed membrane was a BLM based on electrical measurements of the membrane protein α-hemolysin, which produces nanopores in BLMs. Unlike the conventional approach for BLM formation based on the droplet contact method, this method provides aqueous surfaces with no organic solvent coating layer. Hence, this method is suitable for producing BLMs that facilitate the direct addition of chemicals into the aqueous phase.
- Published
- 2021
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175. A Lipid-Bilayer-On-A-Cup Device for Pumpless Sample Exchange.
- Author
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Ito Y, Izawa Y, Osaki T, Kamiya K, Misawa N, Fujii S, Mimura H, Miki N, and Takeuchi S
- Abstract
Lipid-bilayer devices have been studied for on-site sensors in the fields of diagnosis, food and environmental monitoring, and safety/security inspection. In this paper, we propose a lipid-bilayer-on-a-cup device for serial sample measurements using a pumpless solution exchange procedure. The device consists of a millimeter-scale cylindrical cup with vertical slits which is designed to steadily hold an aqueous solution and exchange the sample by simply fusing and splitting the solution with an external solution. The slit design was experimentally determined by the capabilities of both the retention and exchange of the solution. Using the optimized slit, a planar lipid bilayer was reconstituted with a nanopore protein at a microaperture allocated to the bottom of the cup, and the device was connected to a portable amplifier. The solution exchangeability was demonstrated by observing the dilution process of a blocker molecule of the nanopore dissolved in the cup. The pumpless solution exchange by the proposed cup-like device presents potential as a lipid-bilayer system for portable sensing applications.
- Published
- 2020
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176. Rapid and Resilient Detection of Toxin Pore Formation Using a Lipid Bilayer Array.
- Author
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Ito Y, Osaki T, Kamiya K, Yamada T, Miki N, and Takeuchi S
- Subjects
- Cell Membrane, Bacterial Toxins, Lipid Bilayers
- Abstract
An artificial cell membrane is applied to study the pore formation mechanisms of bacterial pore-forming toxins for therapeutic applications. Electrical monitoring of ionic current across the membrane provides information on the pore formation process of toxins at the single pore level, as well as the pore characteristics such as dimensions and ionic selectivity. However, the efficiency of pore formation detection largely depends on the encounter probability of toxin to the membrane and the fragility of the membrane. This study presents a bilayer lipid membrane array that parallelizes 4 or 16 sets of sensing elements composed of pairs of a membrane and a series electrical resistor. The series resistor prevents current overflow attributed to membrane rupture, and enables current monitoring of the parallelized membranes with a single detector. The array system shortens detection time of a pore-forming protein and improves temporal stability. The current signature represents the states of pore formation and rupture at respective membranes. The developed system will help in understanding the toxic activity of pore-forming toxins., (© 2020 Wiley-VCH GmbH.)
- Published
- 2020
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177. Perfusion Chamber for Observing a Liposome-Based Cell Model Prepared by a Water-in-Oil Emulsion Transfer Method.
- Author
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Sugiyama H, Osaki T, Takeuchi S, and Toyota T
- Abstract
For the construction of a chemical model of contemporary living cells, the so-called water-in-oil emulsion transfer (WOET) method has drawn much attention as one of the promising preparation protocols for cell-sized liposomes encapsulating macromolecules and even micrometer-sized colloidal particles in high yields. Combining the throughput and accuracy of the observation is the key to developing a synthetic approach based on the liposomes prepared by the WOET method. Recent advances in microfluidic technology can provide a solution. By means of surface modification of a poly(dimethylsiloxane)-type microfluidic device integrating size-sorting and trapping modules, here, we enabled a simultaneous direct observation of the liposomes with a narrow size distribution, which were prepared by the WOET method. As a demonstration, we evaluated the variance of encapsulation of polystyrene colloidal particles and water permeability of the cell-sized liposomes prepared by the WOET method in the device. Since the liposomes prepared by the WOET method are useful for constructing cell models with an easy protocol, the current system will lead to a critical development of not only supramolecular chemistry and soft matter physics but also synthetic biology., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)
- Published
- 2020
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178. Hydrodynamic accumulation of small molecules and ions into cell-sized liposomes against a concentration gradient.
- Author
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Sugiyama H, Osaki T, Takeuchi S, and Toyota T
- Abstract
In investigations of the emergence of protocells at the origin of life, repeatable and continuous supply of molecules and ions into the closed lipid bilayer membrane (liposome) is one of the fundamental challenges. Demonstrating an abiotic process to accumulate substances into preformed liposomes against the concentration gradient can provide a clue. Here we show that, without proteins, cell-sized liposomes under hydrodynamic environment repeatedly permeate small molecules and ions, including an analogue of adenosine triphosphate, even against the concentration gradient. The mechanism underlying this accumulation of the molecules and ions is shown to involve their unique partitioning at the liposomal membrane under forced external flow in a constrained space. This abiotic mechanism to accumulate substances inside of the liposomal compartment without light could provide an energetically up-hill process for protocells as a critical step toward the contemporary cells., (© 2020. The Author(s).)
- Published
- 2020
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179. A pumpless solution exchange system for nanopore sensors.
- Author
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Yamada T, Kamiya K, Osaki T, and Takeuchi S
- Abstract
This paper proposes a nanopore-based sensor exploiting the solution exchange of a droplet-based lipid bilayer driven by a superabsorbent polymer. Biological nanopores are candidates for use in portable sensors because of their potential to recognize and detect single molecules. One of the current challenges in the development of portable nanopore sensors is the inability to achieve continuous detection. To achieve continuous detection, we have exploited the suction force of a superabsorbent polymer to drive the continuous microfluidic flow required to wash the analyte out of the droplet. The superabsorbent polymer drives the microfluidic flow without electricity, and the developed solution exchange system remains compact. To demonstrate solution exchange in the droplet containing the lipid bilayer, the concentration of heptakis(6- O -sulfo)-β-cyclodextrin was monitored in a time-dependent manner using α-hemolysin nanopores. A reduction in the concentration, attributable to solution exchange, was successfully observed. We believe that the proposed system will increase the portability and usability of nanopore sensors., (Copyright © 2019 Author(s).)
- Published
- 2019
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180. Formation of vesicles-in-a-vesicle with asymmetric lipid components using a pulsed-jet flow method.
- Author
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Kamiya K, Osaki T, and Takeuchi S
- Abstract
Lipid distribution in intracellular vesicles is different from that in the plasma membrane of eukaryotic cells. The lipid components in the intracellular vesicles are composed of phosphatidylserine and phosphatidylethanolamine in the outer leaflet and phosphatidylcholine and sphingomyelin in the inner leaflet. The lipid asymmetricities both in the intracellular vesicle membrane and the plasma membrane contribute to synaptic transmission functions. In this study, we developed a cell-sized asymmetric lipid vesicle system containing small-sized asymmetric lipid vesicles (of diameter 200-1000 nm) (asymmetric vesicles-in-a-vesicle), emulating lipid components in the plasma membrane and intracellular vesicle membrane of eukaryotic cells, using microfluidic technology. We successfully constructed an artificial exocytosis system using the asymmetric vesicles-in-a-vesicle system. This asymmetric vesicles-in-a-vesicle system will be helpful in understanding the mechanisms of vesicle transport, such as neurotransmission and exocytosis., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2019
- Full Text
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181. Construction of a Biohybrid Odorant Sensor Using Biological Olfactory Receptors Embedded into Bilayer Lipid Membrane on a Chip.
- Author
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Misawa N, Fujii S, Kamiya K, Osaki T, Takaku T, Takahashi Y, and Takeuchi S
- Subjects
- Animals, Electrodes, Equipment Design, Octanols analysis, Robotics, Sf9 Cells, Spodoptera, Biosensing Techniques instrumentation, Cell Membrane metabolism, Lipid Bilayers metabolism, Odorants analysis, Receptors, Odorant metabolism
- Abstract
This paper describes an odorant sensor based on mosquito olfactory receptors (ORs) that is sensitive to the volatile organic compound octenol. The ORs and OR coreceptors were reconstructed in the lipid bilayer membrane in a chamber device equipped with electrodes. Using this odorant sensor, we obtained ion current changes caused by specific OR responses to octenol. We installed the odorant sensor into a mobile robot and succeeded in the demonstration of coupling octenol gas detection and robot actuation. We believe that this biohybrid odorant sensing system will be a key technology for future artificial olfaction.
- Published
- 2019
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182. Electrophysiological measurement of ion channels on plasma/organelle membranes using an on-chip lipid bilayer system.
- Author
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Kamiya K, Osaki T, Nakao K, Kawano R, Fujii S, Misawa N, Hayakawa M, and Takeuchi S
- Subjects
- Patch-Clamp Techniques, Ion Channels metabolism, Lab-On-A-Chip Devices, Lipid Bilayers metabolism, Organelles metabolism
- Abstract
Ion channels are located in plasma membranes as well as on mitochondrial, lysosomal, and endoplasmic reticulum membranes. They play a critical role in physiology and drug targeting. It is particularly challenging to measure the current mediated by ion channels in the lysosomal and the endoplasmic reticulum membranes using the conventional patch clamp method. In this study, we show that our proposed device is applicable for an electrophysiological measurement of various types of ion channel in plasma and organelle membranes. We designed an on-chip device that can form multiple electrical contacts with a measurement system when placed on a mount system. Using crude cell membranes containing ion channels extracted from cultured cells without detergents, we detected open/close signals of the hERG, TRPV1, and NMDA channels on plasma membranes, those of the TRPML1 channels on lysosomal membranes, and open/close signals of the RyR channels on SR membranes. This method will provide a highly versatile drug screening system for ion channels expressed by various cell membranes, including plasma, SR, mitochondrial, Golgi, and lysosomal membranes.
- Published
- 2018
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183. Purification-Free MicroRNA Detection by Using Magnetically Immobilized Nanopores on Liposome Membrane.
- Author
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Fujii S, Kamiya K, Osaki T, Misawa N, Hayakawa M, and Takeuchi S
- Subjects
- Humans, MicroRNAs chemistry, Liposomes, Magnetics, Membranes, Artificial, MicroRNAs isolation & purification, Nanopores
- Abstract
MicroRNAs have critical roles in a number of serious diseases and, as a result, are of major interest as clinical diagnostic targets. Conventionally, microRNAs are collected from blood and urine samples and are measured by either quantitative reverse-transcription polymerase chain reaction or microarray. Recently, nanopore sensing techniques have been applied for measuring microRNAs at the single-molecule level. However, existing techniques are technically complex, needing several tools and requiring purification and/or labeling of microRNA samples prior to use. Here we report a method for microRNA detection in a simple procedure requiring neither purification nor labeling. This system utilizes magnetic beads anchored with DNA and nanopores on a liposome membrane. In the presence of the target microRNA, it forms a duplex with complementary DNA, which is then cleaved by a duplex-specific nuclease (DSN). The cleaved DNA, which harbors a liposome on its terminus, is subsequently released from the magnetic bead, fuses to the lipid bilayer on chip, and emits an electrical signal derived from the formation of a nanopore. Because of a property of the DSN, the signals derived from microRNAs are expected to be amplified in an isothermal reaction. Our system possesses the specificity to detect target microRNAs from mixtures containing >10
6 different microRNA sequences and readily uses blood or urine samples. Although the limit of detection is above 10 nM and needs to be improved for practical diagnosis, because purification and labeling are not required, the presented system proposes a possible schematic for the development of easy and on-site diagnosis.- Published
- 2018
- Full Text
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184. Automatic Planar Asymmetric Lipid Bilayer Membrane Formation toward Biological High-Throughput Assay.
- Author
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Gotanda M, Kamiya K, Osaki T, Fujii S, Misawa N, Miki N, and Takeuchi S
- Subjects
- High-Throughput Screening Assays, Lab-On-A-Chip Devices, Lipid Bilayers
- Abstract
This paper describes automation of planar lipid bilayer formation by introducing a stepping motor to a microfluidic device. Planar lipid bilayers or lipid vesicles are useful to understand biological reactions and to investigate the interaction between lipids and proteins. Therefore, to acquire large amount of the information, high-throughput production of planar lipid bilayers or giant vesicles (GVs) is necessary. The droplet split-and-contact method, which enhances the efficiencies of both planar lipid bilayer formation and GV generation, needs to be automated for increasing the throughput. Previous droplet split-and-contact devices were manipulated manually; hence, the influence of manipulation on planar lipid bilayer formation was not evaluated quantitatively. First, to develop an automated system for generating asymmetric planar lipid bilayers, a stepping motor, which allows to control the angular speed of the rotor, is integrated into the droplet split- and-contact device (Fig. $1(\mathrm{b)$). Next, we assessed planar lipid bilayer generation at various angular speeds and found the speed limit for bilayer formation. Finally, we generated asymmetric planar lipid bilayers that have different lipid composition on outer and inner leaflets using this automated device and confirmed the asymmetry of the planar lipid bilayers by generating GVs.
- Published
- 2018
- Full Text
- View/download PDF
185. Stability of the microdroplets for portable biosensor.
- Author
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Izawa Y, Osaki T, Kamiya K, Fujii S, Miki N, and Takeuchi S
- Subjects
- Lab-On-A-Chip Devices, Vibration, Biosensing Techniques
- Abstract
This paper highlights the behavior of microdroplets used for portable biosensors. Microdroplets have been applied for various Lab-on-a-chip applications, in which portability/wearable is becoming a trend word. However, the resonance (sloshing phenomena) of the microdroplets could be an issue. For example, bilayer lipid membrane, fragile characteristics due to its thickness of 5 nm, easily ruptures by jiggling. We therefore prepared various dimensions of droplets by changing the well shape, and examined the resulting waves depending on the vibration frequency between 20 and 100 Hz, including daily life frequencies. Moreover, we investigated an influence of surface coating of the wells by the same tests. The results of the first examination showed that the sloshing was effectively suppressed by reducing the well length along the vibration axis. We also found that the sloshing was suppressed by reducing surface energy of the microwells. We succeeded in clarifying the characteristics of microdroplets at the vibration in daily life frequency, which will be useful information for development of portable biosensors.
- Published
- 2016
- Full Text
- View/download PDF
186. Integrated Microfluidic System for Size-Based Selection and Trapping of Giant Vesicles.
- Author
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Kazayama Y, Teshima T, Osaki T, Takeuchi S, and Toyota T
- Subjects
- Hydrodynamics, Particle Size, Surface Properties, Liposomes analysis, Liposomes chemistry, Microfluidic Analytical Techniques instrumentation
- Abstract
Vesicles composed of phospholipids (liposomes) have attracted interest as artificial cell models and have been widely studied to explore lipid-lipid and lipid-protein interactions. However, the size dispersity of liposomes prepared by conventional methods was a major problem that inhibited their use in high-throughput analyses based on monodisperse liposomes. In this study, we developed an integrative microfluidic device that enables both the size-based selection and trapping of liposomes. This device consists of hydrodynamic selection and trapping channels in series, which made it possible to successfully produce an array of more than 60 monodisperse liposomes from a polydisperse liposome suspension with a narrow size distribution (the coefficient of variation was less than 12%). We successfully observed a size-dependent response of the liposomes to sequential osmotic stimuli, which had not clarified so far, by using this device. Our device will be a powerful tool to facilitate the statistical analysis of liposome dynamics.
- Published
- 2016
- Full Text
- View/download PDF
187. Round-tip dielectrophoresis-based tweezers for single micro-object manipulation.
- Author
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Kodama T, Osaki T, Kawano R, Kamiya K, Miki N, and Takeuchi S
- Subjects
- Cell Tracking, Humans, Single-Cell Analysis, Staining and Labeling, Gold chemistry, Liposomes chemistry, Micromanipulation instrumentation, Micromanipulation methods, Optical Tweezers
- Abstract
In this paper, we present an efficient methodology to manipulate a single micro-object using round-tip positive dielectrophoresis-based tweezers. The tweezers consist of a glass needle with a round-tip and a pair of thin gold-film electrodes. The round-tip, which has a radius of 3µm, is formed by melting a finely pulled glass needle and concentrates the electric field at the tip of the tweezers, which allows the individual manipulation of single micro-objects. The tweezers successfully captured, conveyed, and positioned single cell-sized liposomes with diameters of 5-23µm, which are difficult to manipulate with conventional manipulation methodologies, such as optical tweezers or glass micropipettes, due to the similarities between their optical properties and those of the media, as well as the ease with which they are deformed or broken. We used Stokes' drag theory to experimentally evaluate the positive dielectrophoresis (pDEP) force generated by the tweezers as a function of the liposome size, the content of the surrounding media, and the applied AC voltage and frequency. The results agreed with the theoretically deduced pDEP force. Finally, we demonstrated the separation of labeled single cells from non-labeled cells with the tweezers. This device can be used as an efficient tool for precisely and individually manipulating biological micro-objects that are typically transparent and flexible., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
188. Nano bioresearch approach by microtechnology.
- Author
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Collard D, Kim SH, Osaki T, Kumemura M, Kim B, Fourmy D, Fujii T, Takeuchi S, Karsten SL, and Fujita H
- Subjects
- Biomedical Research methods, DNA chemistry, Lipid Bilayers chemistry, Microtechnology methods, Nanotechnology methods
- Abstract
To progress in basic science and drug development, convenient methodology for detecting specific biological molecules and their interaction in living organism is in high demand. After more than 20 years of increasing research efforts, micro and nanotechnologies are now mature to propose a new class of miniature devices and principles enabling compartmentalized bioassays. Among them, this review proposes various examples that include array of electro-active microwells for highly parallel single cell analysis, cost-effective nanofluidic for DNA separation, parallel enzymatic reaction in 100pL droplet and high-throughput platform for membrane proteins assays. The micro devices are presented with relevant experiments to foresee their future contribution to translational research and drug discovery., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
189. Multichannel simultaneous measurements of single-molecule translocation in alpha-hemolysin nanopore array.
- Author
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Osaki T, Suzuki H, Le Pioufle B, and Takeuchi S
- Subjects
- Lipid Bilayers chemistry, Microfluidic Analytical Techniques instrumentation, Bacterial Toxins chemistry, DNA, Single-Stranded chemistry, Hemolysin Proteins chemistry, Microfluidic Analytical Techniques methods, Nanostructures chemistry, Poly U chemistry
- Abstract
We present a microarray system that enables simultaneous monitoring of multiple ionic currents through transmembrane alpha-hemolysin nanopores arrayed at bilayer lipid membranes. We applied the self-assembling ability of lipid molecules interfaced between an aqueous solution and organic solvent to induce bilayer membrane formation at a microfluidic device; the device consists of a hydrophobic polymer film that serves to suspend the lipid-containing solvent at micrometer-sized apertures as well as to separate the aqueous solution into two chambers. In this study, we confirmed that expeditious and reproducible bilayer formation is realized by control of the composition of the solvent, a mixture of n-decane and 1-hexanol, which permits simultaneous incorporation of the alpha-hemolysin nanopores to the membrane array. Monitoring the eight wells on the array at once, we obtained a maximum of four relevant, synchronous signals of translocating ionic current through the nanopores. The system was also able to detect translocation events of nucleic acid molecules through the pore via the profile of a blocked current, promising its potential for high-throughput applications.
- Published
- 2009
- Full Text
- View/download PDF
190. Combined microslit electrokinetic measurements and reflectometric interference spectroscopy to study protein adsorption processes.
- Author
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Zimmermann R, Osaki T, Gauglitz G, and Werner C
- Abstract
Streaming potential/current measurements for the characterization of charge formation processes at solid/liquid interfaces were combined with reflectometric interference spectroscopy. The simultaneous determination of electrosurface characteristics and the optical thickness of interfacial layers provides information on structural variations of adsorbed or covalently bound polymers and on charge dependent adsorption and desorption phenomena at solid/liquid interfaces. To demonstrate the potentialities of this extended approach for biointerfacial studies the authors report a series of experiments on the adsorption of the plasma protein fibrinogen at poly(octadecene-alt-maleic acid) thin films., (2007 American Vacuum Society.)
- Published
- 2007
- Full Text
- View/download PDF
191. Electrostatic switching of biopolymer layers. Insights from combined electrokinetics and reflectometric interference.
- Author
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Zimmermann R, Osaki T, Kratzmüller T, Gauglitz G, Dukhin SS, and Werner C
- Subjects
- Electric Conductivity, Electrochemistry, Hydrogen-Ion Concentration, Static Electricity, Biopolymers chemistry, Spectrum Analysis methods
- Abstract
Structural integrity and functional characteristics of biomacromolecules are largely defined by electrostatic forces between ionized moieties, which are often altered at interfaces. Unraveling these changes requires access to charge state and structure of surface-confined biopolymers in aqueous environments. We therefore combined electrokinetic measurements of interfacial electrical potentials with the simultaneous determination of the optical layer thickness by reflectometric interference spectroscopy. Two examples are summarized to demonstrate the resulting options: The pH-switching of grafted poly(l-glutamic acid) layers caused by dissociation-dependent helix-coil transitions was studied; potential distribution and ion mobility within the grafted polyelectrolyte were unraveled using an new theoretical model for the charging of polyelectrolyte layers. The charge-driven modulation of biopolymers at interfaces was furthermore analyzed in the adsorption of fibronectin onto polymer substrates with varied charge density; the results permit us to reach a conclusion about the relevance of electrostatic matching for orientation and anchorage of the protein. Altogether, the introduced methodology was found suitable to follow the electrosurface characteristics of biomacromolecules in situ.
- Published
- 2006
- Full Text
- View/download PDF
192. Hydrophobic and electrostatic interactions in the adsorption of fibronectin at maleic acid copolymer films.
- Author
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Osaki T, Renner L, Herklotz M, and Werner C
- Subjects
- Adsorption, Static Electricity, Fibronectins chemistry, Maleates chemistry, Polymers chemistry
- Abstract
Adsorption and desorption of fibronectin (FN) were investigated at thin films of alternating maleic acid copolymers with octadecene (POMA) and with propene (PPMA). The hydrophobicity and charge density of the polymers were modulated by the choice of the comonomer. In consequence, the dominant forces between the substrate and the protein were specified as hydrophobic interaction for POMA and electrostatic interaction for PPMA. The adsorption kinetics were investigated in situ as variations of the optical thickness, adsorbed mass, and viscoelastic properties (detected by reflectometric interference spectroscopy and quartz crystal microbalance technique, respectively) while alterations of the electrosurface properties were derived from surface conductivity data and isoelectric points (by streaming potential/current measurements using a microslit electrokinetic setup). The results demonstrate that the interfacial mode of adsorbed FN depends on the predominant interactions: large amounts of FN were tightly bound to POMA by hydrophobic interactions. In contrast, FN adsorbed on PPMA was concluded to attain an unfolded structure allowing for the "electrostatic matching" of positively charged residues on FN with the maleic acid groups. This conclusion was supported by the acidic IEP of 3.2 found for FN on PPMA and a significant reduction of the surface conductivity of the FN-covered polymer film, whereas FN on POMA showed an IEP of 4.2 (close to the intrinsic IEP of FN), indicating a stochastic orientation of the adsorbed protein.
- Published
- 2006
- Full Text
- View/download PDF
193. Polyanion protection of silane bonds to silicon oxide revealed by electrokinetic measurements.
- Author
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Osaki T, Zimmermann R, Kratzmüller T, Schweiss R, and Werner C
- Published
- 2004
- Full Text
- View/download PDF
194. Ionic Environmental Effect on the Time-Dependent Characteristics of Membrane Potential in a Bipolar Membrane.
- Author
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Suendo V, Eto R, Osaki T, Higa M, and Tanioka A
- Abstract
The membrane potential characteristics of a bipolar membrane are discussed in an examination of the contribution of an intermediate phase to time-dependent behavior. Bipolar membranes, which consist of a poly(sulfone) base polymer with a quaternary amino group and a poly(styrene-co-divinylbenzene) cation-exchange membrane, were prepared in this study. The membrane potentials in various external concentration differences, the facing directions of the membrane, and the external electrolyte solutions were measured as a function of time. In this study, the time course of the membrane potential was simulated by using the equation based on the Teorell-Meyer-Sievers theory and by assuming the concentration in the intermediate phase, which is much higher or lower than that in the external solution. The membrane facing direction and the intermediate phase condition will alter the direction of the membrane potential change. The intermediate phase in a bipolar membrane seems to act as an alteration barrier for the membrane potential according to the membrane facing direction. Copyright 2001 Academic Press.
- Published
- 2001
- Full Text
- View/download PDF
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